Research Experiences for Undergraduates (REU) Participants-Department of Biological Sciences - Carnegie Mellon University

2010 Participants

National Science Foundation Research Experiences for Undergraduates (REU)

Undergraduate Research Experiences in Cellular and Molecular Biosciences

Hossam AbdouHossam Abdou, Pennsylvania State University

Mentor: Dr. Jonathan Minden

Developing an Effective Marker for Autophagy in Live Organisms

The Minden lab studies apoptosis and the clearance of apoptotic cells. Apoptosis is necessary for an organism’s normal development, homeostasis, and for the clearance of potentially cancerous cells. Dr. Elane Fishilevich, a postdoc in the Minden lab, developed a biomarker to observe the engulfment of apoptotic cells in live, developing organisms. Recently, they observed possible interactions between apoptosis and autophagy. A biomarker for autophagy will enable us to visualize this process in live embryos and will help resolve the nature of the relationship between autophagy and apoptosis in a developmental context. A biomarker, pHanTom, has been developed for this purpose. pHanTom comprises two parts: pHluorin, a pH-sensitive fluorescent protein, and Tom20, which localizes pHanTom to mitochondria. After an unwanted mitochondrion has been consumed by an autophagosome, via autophagy, and the autophagosome has fused with the lysosome, the discarded mitochondrion is exposed to the acidic pH of the lysosome. The mitochondria-localized pHluorin should indicate this pH drop and thus, autophagy. I used standard cloning techniques to make DNA constructs, and have initiated the process that will ultimately result in the generation of Drosophila cell lines that stably express pHanTom. Imaging has shown that pHanTom is localized correctly in both transiently and stably transfected cells. To determine the effectiveness of pHanTom as a biomarker of autophagy, transiently-transfected cells were incubated with a drug known as Rapamycin to induce autophagy and imaged. Two issues with regard to this imaging had to be resolved. First, excessive light exposure was killing the pHanTom cells, so we determined how much light exposure pHanTom cells could withstand without dying. Second, Rapamycin was killing the cells at high concentrations, so we determined how much could be used to induce autophagy without killing the cells. Once the proper settings and concentrations were determined, the cells were imaged to determine the ability of pHanTom to serve as a biomarker for autophagy.

Krista Carter

Krista Carter, Marietta Collge

Mentor: Dr. Brooke McCartney

Testing the Role of Phosphorylation and Salt Bridge Interactions Between Drosophila APC2 and Armadillo in Wnt Signaling

Adenomatous polyposis coli (APC) is a tumor suppressor that is mutated in cases of colon cancer and is a negative regulator of the Wnt signaling pathway. It functions with other proteins as part of a destruction complex that binds to and leads to the degradation of β-catenin, a key effector of Wnt signaling. Biochemical analysis suggests that phosphorylation of APC and salt bridge interactions between APC and β-catenin are important in the function of the destruction complex. To test this in vivo, the lab has generated different Drosophila APC2 mutants that affect phosphorylation and salt bridge interactions. Transgenic lines that express these mutant APC2 proteins have also been established. To assay the function of the mutant APC2 proteins, we have determined their ability to rescue defects associated with the complete loss of APC2 in the Drosophila embryo. Loss of endogenous APC2 function in the embryo leads to lethality, cuticle defects, and increased levels of Armadillo (Drosophila equivalent of β-catenin) as a result of increased Wnt signaling. Previous analysis of these defects has shown that the APC2 mutants are able to rescue most normal APC2 function and suggests that phosphorylation and salt bridge interactions are not essential for the destruction complex. In addition to this analysis, I have further analyzed the APC2 mutants by using immunostaining and confocal microscopy to look at the expression of Engrailed, a transcriptional target of Wnt signaling. Engrailed expression is increased in APC2 mutants due to the elevation of Wnt signaling. The phosphorylation mutants rescued Engrailed expression somewhat better than the salt bridge mutants. Consistent with previous analysis, the loss of phosphorylation and salt bridges does not completely disrupt the function of the destruction complex. However, the salt bridges may play a more important role than phosphorylation in binding APC2 and Armadillo in the destruction complex.

Justin ChenJustin Chen, Oberlin College

Mentor: Dr. Chuck Ettensohn

Exploring the Function of Duplicated Genes in the Evolution of an Embryonic Cell Lineage

Primary mesenchyme cells (PMCs) are the best characterized cell population in the sea urchin embryo. During embryonic development, PMCs undergo a remarkable sequence of morphogenetic events, highlighted by a migration from the vegetal pole of the blastocoel to form a characteristic ring like arrangement in the gastrula, that culminates in the formation of the larval skeleton. Genes within the gene regulatory network responsible for skeletogenesis or PMC differentiation have duplicate forms. Two such duplicated pairs are alx1 and alx1-like as well as lasp1 and lasp1-like. Both alx1 and lasp1 are expressed exclusively in PMCs. Alx1 regulates genes responsible for the epithelial to mesenchyme transition in PMCs and biomineralization (skeleton formation). Although lasp1 , a cytoskeletal scaffold protein associated with human breast cancer, has not been studied extensively in sea urchins, it could play a role in PMC migration. Gene duplication is a process that frees the second copy of the gene from selection pressure allowing it to accumulate mutations at a faster rate than normal and acquire novel functions. In general, duplicated genes have a more specific function than the original gene. In this study, whole mount in situ hybridization was used to determine expression patterns of alx1-like and lasp1-like. The location and timing of gene expression allows the researcher to make hypotheses regarding basic gene function and lays a foundation for future experiments in which gene function is tested directly. Preliminary results indicate that alx1-like is specific to PMCs, suggesting that the duplicated gene still plays a role in PMC development. In contrast, lasp1-like is expressed in the majority of embryonic cell types, demonstrating that lasp1-like has a more general role in sea urchin development.

Jeremy Gale

Jeremy Gale, The Richard Stockton College of NJ

Mentor: Dr. Veronica Hinman

MicroRNA Expression and Function in Echinoderm Development

microRNAs (miRNAs) are single stranded non-coding RNAs 21-25 nucleotides in length that act as posttranscriptional repressors of gene expression. Since their discovery in 1998, miRNAs have been found to be evolutionarily conserved amongst eukaryotes, and evidence suggests that they play a major role in both development and cell differentiation. Although microRNAs are necessary for the correct development of many animals, their role in echinoderm embryogenesis has yet to be characterized. To understand the role of miRNAs in echinoderm embryos, the developmental expression and function of these genes was analyzed. The spatial expression of miRNA 31 (miR-31), a gene abundant in embryos of the starfish, Pateria miniata, was examined. This miRNA shows restricted expression in certain stages of embryogenesis, which implicates it in ectoderm development. To determine the role of miRNAs in sea urchin development, the function of the enzyme Dicer was blocked in Strongylocentrotus purpuratus to prevent synthesis of mature miRNAs. Expression of genes involved in the differentiation of endomesoderm, cilia, pigment cells, and skeletogenic mesoderm were analyzed in such embryos and a reproducible increase in expression of enzymes required for pigment synthesis was observed. This study suggests that miRNAs are involved in echinoderm embryogenesis and may function in the differentiation of particular cell types.

Daniel Gerber

Daniel Gerber, Carthage College

Mentor: Dr. Nathan Urban

Neuron Variability and Stochastic Synchrony in the Olfactory Bulb

The brain is made up of many different types of neurons. For example, the olfactory bulb contains two different main neuronal types: excitatory mitral cells and inhibitory granule cells. In computer simulations of neuronal networks, it is common to treat all of the neurons of a given type as identical; however, this is not the case in a real brain. As Dr. Urban’s lab has previously shown, there are specific intrinsic properties that affect the performance of a neuron, and these properties are variable from neuron to neuron. Specifically, these properties can affect the level of synchrony between mitral cells in response to a stochastic (random white noise) stimulus. Stochastic synchrony has been found in multiple areas of the brain including the olfactory bulb, and has been shown to play an important role in certain brain disorders, such as Parkinson’s Disease and autism. This summer, I worked on quantifying the effects that variations in a neuron’s Phase Resetting Curve (PRC) have on its synchronization with other neurons. The Phase Resetting Curve is an intrinsic neuronal quality that defines how much a stimulus will change a neuron’s state at different times. To accomplish this, I used data collected by the lab from mouse mitral cells to parameterize actual PRCs and to determine the amount of PRC heterogeneity in a neuronal population. Once an effective parameterization was created, it was used to simulate large neuron populations and to explore their synchronization. I determined that the synchronization of a neuron pair is correlated with the difference between their PRCs, and that similarity in the magnitude parameter of the PRCs is more influential to synchrony than either the shape parameter or the flatness parameter.

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Jasmin Kaeser, Centre College

Mentor: Dr. Russell Schwartz

Unmixing Population Genetics Data

While there are several methods currently available to study population ancestry, new models may help increase the accuracy of findings and decrease the amount of time it takes to determine ancestry. Recent work using population genetics tools to look at tumor cell growth resulted in the adoption of a new method of data interpretation known as unmixing. This method was used in tumor research to determine ancestral cell states in tumor evolution and may have similar applications for human populations of mixed ancestry. The purpose of this research is to determine whether unmixing is a valid tool for looking at single nucleotide polymorphisms (SNPs) to determine the ancestry of populations and to maximize the usefulness of this method for population genetics. In order to accomplish this, we used MatLab to test data gathered by the HapMap Project, an international survey of human genetic variation, and simulated populations created with ms, a population simulator program. We looked at the effectiveness of unmixing under different parameters such as number of individuals included and the amount of SNPs, as well as with different admixed populations with multiple ancestries. We found that the standard deviation around the central point of ancestry increased as the number of SNPs analyzed increased, but found no similar correlation with the number of individuals analyzed. We also found that unmixing handles three pure populations best, but still has a low error rate when given data with two populations admixed together. It does best with smaller generation sizes, with error increasing with generation size. If two admixed populations are analyzed together this increase is less linear, but the error size is only slightly higher overall. So unmixing can handle admixed populations, but further research is needed to see to what extent this is true.

Rosalie Lawrence

Rosalie Lawrence, Swarthmore College

Mentor: Dr. David Hackney

Ectopic Expression of Mitochondrial-Tagged Kinesin Constructs and Distribution of Mitochondria in HeLa Cells

Kinesin-1 is known to transport cargos toward the positive end of microtubules, located at the periphery of the cell. Transport of cargos, such as mitochondria, is necessary to allow for organelle localization to facilitate vital cellular processes such as action potentials. While it is known that cargos are typically bound to multiple motor types in response to stimuli and cellular needs, regulatory mechanisms for determining the direction of transport are not yet fully understood. In order to gain insight into the role Kinesin-1 plays in the more complex system of organelle organization and transport, fluorescently-tagged Drosophila melanogaster and Homo sapiens kinesin constructs including the C terminus of the ActA gene, which codes for a protein with a specific binding site to the mitochondrial outer membrane, were exogenously expressed in HeLa cells via the vector pEGFP-C1. Increasing exogenous Kinesin-1 levels was expected to increase mitochondrial processivity toward the plus end of microtubules. Furthermore, we expected increased processivity for constructs lacking Kinesin-1 tail domains, which have been shown to regulate binding, relative to full-length constructs. Preliminary results suggest that exogenously expressing the Drosophila Kin1 construct lacking tail domains causes mitochondrial localization at the periphery of the cell. These results support the theory that increasing the number of bound kinesins on mitochondria may overwhelm negatively-directed motors, and result in net positive-directed motion.

Kathleen Maher

Kathleen Maher, St. Vincent College

Mentor: Dr. Javier Lopez

Imaging of Fluorescent DNA Nanotags for RNA Localization

Subcellular RNA localization is an important mechanism that ensures that proteins are targeted to the area of the cell in which they are needed. Drosophila is a valuable model system in which to identify the responsible factors and identify their mode of action. During the syncytial stage of Drosophila embryos the nuclei divide many times before cytokinesis, so that polarization and development are contingent on the correct localization of many maternal and zygotic mRNAs. Although several RNA imaging techniques exist, improvements are required for dynamic studies in live embryos, such as maintaining low background fluorescence, improving specificity, and amplifying the signal to detect mRNAs expressed in low amounts. In this project DNA nanotags generated by the Armitage lab will be used to image RNA localization in Drosophila melanogaster. The system combines a nanotag probe composed of DNA with YOYO donor dyes and a separate probe with a terminal Cy-5 acceptor. The two probes target adjacent regions on a specific mRNA so as to emit a FRET signal when both components hybridize with the RNA. The DNA nanotags must be characterized to ensure that they are functioning properly. I tested nanotag viability to determine the lifetime of the tags and to optimize delivery concentrations. I then transfected the DNA nanotags into Drosophila Schneider 2 cells (S2) with oligofectamine and microinjected into syncytial Drosophila embryos. The nanotags transfect very efficiently into the S2 cells. Also, when microinjected into Drosophila embryos, the nanotags dispersed well and were subsequently found in every cell. The nanotags did not exhibit toxicity; the injected embryos continued to develop normally and hatched as viable larvae. In both cases, FRET signaling was observed, indicating that the nanotags were not being degraded. In the transfection, the FRET signaling could be detected up to 120 hours post-transfection.

Christabel Okegbe

Christabel Okegbe, Sewanee: The University of the South

Mentor: Dr. David Hackney

Tracking Ectopically-Expressed Kinesin Powered Transport of Golgi Apparatus within HeLa Cells

The movement of organelles like golgi within the cell is paramount to cellular function. Motor proteins such as the kinesin family members are responsible for transporting these organelles. Kinesin is a protein hetero-tetramer with two heavy and two light chains. The heavy chain contains a motor domain, which is connected to a coiled-coil stalk, and culminates in a tail region. Vesicles bind to the light chain. Kinesins have two separate binding sites for microtubules and ATP on each head, and move along microtubules driven by the hydrolysis of ATP via a head-over-head mechanism. They generally carry out anterograde transport i.e. conveying cargo to the periphery of the cell. Our research focused on wild-type Drosophila kinesin-1 up to amino acid residue 558 and Human kinesin-1 up to residue 541, which we generated using PCR and cross-checked using sequencing primers. We used kanamycin resistant pEGFP-C1 into which kinesin, GFP, and part of the C-terminal region of the golgi membrane protein known as Giantin were inserted at specific restriction sites, to later on express the desired Kinesin-GFP-Giantin (KGG) protein for both fly and human kinesin. The plasmid construct was reproduced via electroporation cells. HeLa cells will be transformed with this new plasmid construct, and the KGG protein will be expressed and targeted to the golgi. The distribution of golgi within the cells will be monitored via fluorescence microscopy. Future work will also involve using other controllable expression systems to switch from low to higher expression levels of kinesin quantifiable by western blot assays, and determining what effect that will have on golgi distribution within the cell. Using other kinds of cells in our study is another potential direction to follow, if existing literature points us to cells that operate with uniquely positioned golgi apparatus.

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